Process for producing fatty acid esters of sugars

ABSTRACT

A process for producing fatty acid esters of sugars by fermentation which comprises culturing a hydrocarbon-assimilating micro-organism under aerobic conditions in an aqueous nutrient medium containing hydrocarbons as the main source of carbon. Good yields are obtained using n-paraffins of six to 25 carbon atoms or kerosene as the hydrocarbon in the medium. Exemplary microorganisms are those belonging to the genus Arthrobacter, Brevibacterium, Micrococcus, Corynebacterium, Mycobacterium, Candida, or Aspergillus.

United States Patent Tanaka et al.

[451 Jan. 25, 1972 [72] Inventors: Katsunobu Tanaka; Takeo Suzuki, bothof Machida-shi, Japan Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan [22]Filed: Jan.31, 1969 [21] Appl.No.: 795,738

[73] Assignee:

[30] Foreign Application Priority Data Feb. 1, 1968 Japan ..43/5735 [52]U.S. Cl. ..l95/28 R [51] Int. Cl. ..C12d 13/00 [58] Field of Search..195/28, 30

[56] References Cited UNITED STATES PATENTS 3,219,543 11/1965 Douros eta1. ..l95/1 3,409,506 11/1968 Stevens et a1. ..195/28 OTHER PUBLICATIONSShaw et al., Nature, Vol. 217, p. 142 January 13. 1968 Tulloch et al.,Canadian J. Chemistry, Vol. 40, p. 1326 (1962) Lennarz, Journal ofBiological Chemistry, Vol. 239, p. PC 31 10 1964.

Anderson et al., Journal of Biological Chemistry, Vol. 101, p 499 (1933)1 Primary ExaminerA. Louis Monacell Assistant ExaminerSeymour RandAttorney-Craig, Antonelli & Hill [5 7] ABSTRACT A process for producingfatty acid esters of sugars by fermentation which comprises culturing ahydrocarbon-assimilating micro-organism under aerobic conditions in anaqueous nutrient medium containing hydrocarbons as the main source ofcarbon. Good yields are obtained using n-paraffins of six to 25 carbonatoms or kerosene as the hydrocarbon in the medium. Exemplarymicro-organisms are those belonging to the genus Arthrobacter,Brevibacterium, Micrococcus, Corynebacterium, M ycobacterium, Candida,or Aspergillus.

15 Claims, No Drawings PROCESS FOR PRODUCING FATTY ACID ESTERS OF SUGARSThis invention relates to a process for producing fatty acid esters ofsugars. More particularly, it relates to a process for the production offatty acid esters of sugars by fermentation. Even more particularly, theinvention relates to a process for producing fatty acid esters of sugarsby fermentation with micro-organisms which are capable of utilizinghydrocarbons.

The present inventors have found that micro-organisms capable of growingby means of the utilization of hydrocarbons generally have surfaceactive substances in the cells or on the surface of the cells thereof.Furthermore, it has been confirmed by extracting and identifying thesesubstances that they are fatty acid esters of various sugars. Esters ofsugars are widely used as surface active agents and in variousindustries. Accordingly, there is a need in the art for a fermentationprocess for producing fatty acid esters of sugars inexpensively and in ashort period of time.

One of the objects of the present invention is to provide a process forproducing fatty acid esters of sugars.

Another object of the present invention is to provide a process forproducing fatty acid esters of sugars by fermentation which may becarried out in an efficacious and relatively simple manner.

A further object of the invention is to provide a process for producingfatty acid esters of sugars by fermentation which may be carried outadvantageously on an industrial scale at low cost to give a high yieldof product.

A still further object of the invention is to provide fatty esters ofsugars.

These and other objects and advantages of the present invention willbecome apparent to those skilled in the art from a consideration of thefollowing specification and claims.

As the result of investigation on the production of various sugar estersby fermentation with the use of micro-organisms from inexpensivestarting materials, the present inventors have found, in accordance withthe present invention, that significant quantities of fatty acid estersof sugars are produced and accumulated in the fermentation liquor whenmicro-organisms capable of utilizing hydrocarbons are cultured in anaqueous nutrient medium under aerobic conditions. A large variety ofmicro-organisms may be employed in the present invention. As notedabove, the micro-organisms should be capable of assimilating andutilizing hydrocarbons. Examples of micro-organisms suitable forcarrying out the present invention are as follows:

Arthrobacter paraffineus Arthrobacter roseoparafiinus Arthrobacterhydrocarboclastus Arthmbacter simplex Brevibacterium ketoglutamicumMicrococcus paraffinolyticus Corynebacterium hydrocarboclastusCorynebacteriumfascians Coryhebacterium pseudodiphtheriticumCorynebacteriumfascians M ycobacterium smegmatis Candida lipolyticaAspergillus oryzae As is clear from the above list, micro-organismswhichproduce and accumulate fatty acid esters of sugars utilizinghydrocarbons as the carbon source exist widely irrespective of genus andfamily.

Either a synthetic culture medium or a natural nutrient medium issuitable for the cultivation of the strains employed in the presentinvention as long as it contains the essential nutrients for the growthof the strain employed. Such nutrients are well known in the art andinclude substances such as a carbon source, a nitrogen source,inorganic'compounds and'the like which are utilized by themicro-organism employed in appropriate amounts.

The fermentation in connection with the present invention is conductedin an aqueous nutrient medium containing a hydrocarbon or a mixture ofhydrocarbons as the main carbon source. Such hydrocarbons includestraight and branchedchain paraffins (alkanes) having from six to 25carbon atoms, such as n-hexane, n-octane, n-decane, n-dodecane,n-hexadecane, n-eicosane, isooctane, etc., cycloparaffins such ascyclohexane and cyclooctane, straightand branched-chain olefins such ashexene-l ocetene-l octene-2, etc., cycloolefins such s cyclohexene,aromatic hydrocarbons such as benzene, the isomeric xylenes, etc., andmixtures thereof andhydrocarbons derived from petroleum such askerosene, light oils, heavy oils, paraffin oils, etc. Small amounts ofother carbon sources such as carbohydrates, for example, glucose,fructose, maltose, sucrose, starch, starch hydrolysate, molasses, etc.,or any other suitable carbon source such as organic acids, for example,acetic acid, lactic acid, etc., may be used in the fermentation mediumalong with the hydrocarbon. These substances may also be used eithersingly or in mixtures of two or more.

As a nitrogen source, various kinds of inorganic or organic salts orcompounds, such as urea, liquid ammonia or ammonium salts such asammonium chloride, ammonium sulfate, ammonium nitrate, ammonium acetate,ammonium phosphate, etc., or natural substances containing nitrogen,such as cornsteep liquor, yeast extract, meat extract, peptone, fishmeal, bouillon, casein hydrolysates, casamino acid, fish solubles, ricebran extract, etc., may be employed. Again, these substamces may be usedeither singly or in combinations of two or more.

Inorganic compoundswhich may be added to the culture medium includemagnesium sulfate, sodium phosphate, potassium dihydrogen phosphate,potassium monohydrogen phosphate, iron sulfate, manganese chloride,calcium chloride, sodium chloride, zinc sulfate, etc.

Moreover, in the case of certain strains, it may also be necessary toadd certain essential nutrients to the culture medium, such as aminoacids, for example, aspartic acid, threonine, methionine, etc., and/orvitamins, for example, biotin, thiamine, cobalamin and the like.substances in conducting culturing in accordance with the presentinvention, the fermentation medium is sterilized and then themicro-organism to be employed is inoculated therein. Culturing isconducted under aerobic conditions, such as aerobic shaking of theculture or with stirring and aeration of a submerged culture, at atemperature of, for example, about 25 to 40 C. and at a pH of, forexample, about 4 to 9. During cultivation, the pH is adjusted to therecited range (preferably 6 8 by adding, for example, urea solution,ammonia water or ammonium carbonate solution to the medium. Fermentationis completed usually in 2 to 4 days. In practice, the fermentation isterminated when the measurement value of total esters shows a maximum.

The product fatty acid esters of sugars accumulate predominately in theoily layer part of the fermentation liquor. Accordingly, after thecompletion of fermentation, the aqueous part of the lower layer of thefermentation liquor is removed by letting the fermentation liquor standstill in a cold room or by using centrifugal separation. Achloroformmethanol (1 l mixture solution is added to the upper layerpart so as to extract the total lipids therefrom. The solvent is removedat 40 C. under reduced pressure and, then, the oily part of the upperlayer is recovered by centrifugal separation. The thus-obtained oillayer is passed through a silica-gel column and is eluted with hexane inorder to remove the nparaffins contained therein. Subsequently, polarcompounds are removed by elution with chloroform. The fatty acid estersof sugars are recovered after conducting elution withchloroform-methanol mixture solution, gathering the respectivefractions, removing the solvent, adding cold acetone gathering theresulting precipitates and finally purifying with acetone.

The following examples are given merely as illustrative of the presentinvention and are not to be considered as limiting. Unless otherwisenoted, the percentages therein and throughout the application are byweight per liter of fermentation liquor. Although particularmicro-organism strains are exemplified therein, it is to be understoodthat any other of the micro-organisms or mutants thereof having theproperties described hereinabove may be used.

EXAMPLE 1 Arthrobacter paraffineus ATCC 15591 is cultured in a seedmedium containing 1.0 percent meat extract, 1.0 percent peptone and 0.3percent sodium chloride, a medium having a pH of 7.2 beforesterilization, with aerobic shaking for 24 hours. The resultant seedculture is inoculated in the ratio of percent by volume into 3.0 litersof a fermentation medium contained in a 5 -liter jar fermentor andhaving the following composition:

Culturing is conducted at 30 C. with agitation at the rate of 600 rpm.and with aeration with sterile air at the rate of one liter per literper minute for 80 hours. At the beginning of culturing, 600 ml. ofamixture ofC C n-paraffins is added to the culture medium, and the pH ofthe medium is adjusted to 6.8 -7.5 with ammonia water. After thecompletion of fermentation, 2.5 liters of the fermentation liquor isseparated by means of a centrifuge, and the water-soluble part of thelower layer is removed by suction. To 0.4 liter of the upper layer part,a fivefold mixture solution of chloroform-methanol (1 l is added. Afterextracting with agitating at room temperature for 30 minutes the upperlayer part is filtered. The resultant precipitates are washed with theabove-mentioned solvent. The filtrate and washed solution are mixed andadjusted to a volume of 3.2 liters. Then, the solvent contained thereinis removed at 40 C. under reduced pressure, and the solution is heatedand separated with a centrifuge. The upper layer is recovered andn-hexane is added thereto. The, the resulting solution is filuted to 250ml. and passed through a silica-gel column (5.2 2O cm.). Subsequently,n-hexane is passed through the column in order to flush out anyremaining nparaffins. Thereafter, chloroform is passed through thecolumn in order to remove nonpolar substances. After gathering thefractions (600 ml. eluted with the chloroformmethanol (95:5) mixturesolution, removing the solvent at 40 C. under reduced pressure, addingwarm acetone to dissolve the mixture and letting it stand still in acold room, a precipitate having a jelly state is recovered. When theprocedure of dissolution with acetone and precipitation is repeatedsimilarly as described above, 5.3 g. ofa pale yellowjellylike substanceis obtained.

As the result of conducting various identification tests, the productobtained is determined to be a mixture of esters having one ester bondper one molecule of glucose and two ester bonds per one molecule oftrehalose with a higher fatty acid (arthronic acid) having a hydroxygroup at B-position and a branching at the a-position. The product hasthe property of being capable of dispersing mixtures of water andmineral oil or water and n-paraffins extremely effectively.

EXAMPLE 2 Corynebaclerium hydrocarboclastus ATCC 15592 is cultured inthe same fermentation medium and under the same conditions as describedin example 1, except that a C -C nparaffin-mixture is used as the carbonsource. Culturing is conducted in a 5-literjar fermentor. After 72 hoursof culturing, 2.3 liters of the resulting fermentation liquor is treatedin the same manner as in example 1. Consequently, 0.65 liter of an oillayer part is obtained. 4.5 liters of a mixture solution ofethanol-ether (2:1) is added thereto and, after mixing suffrciently, thesolution is filtered. 5.1 liters of the orange-yellow extractionsolution is heated to 40 C. under reduced pressure so as to remove thesolvent therefrom and, after the residual solution is heated, thetransparent solution of the upper layer is recovered by centrifugalseparation. Subsequently, a column chromatography procedure is conductedas described in example 1. As a result, 3.6 g. of an ester of trehalosewith a fatty acid having an experimental formula of C H O, is separatedas a single substance. In addition, 0.2 g. of a fatty acid ester ofglucose is separated.

EXAMPLE 3 Micrococcus epidermidis ATCC is cultured in a culture mediumcontaining 2 percent sorbitol, 1 percent peptone, 1 percent meat extractand 0.3 percent sodium chloride with aerobic shaking for 24 hours usingan n-paraffin mixture containing C, C,:, fractions as a carbon source inthe same manner as described in example 1. The resultant seed culture isinoculated in the ratio of 10 percent by volume into a fermentationmedium having the following composition:

10% kerosene l mg.ll. CuSO,-5H,O 1.0% (NH,),SO, 0.1% cornstecp liquor 207]. thiamine The pH thereof is adjusted to 7.5 (before sterilization).

Culturing is carried out with aerobic shaking of the culture at 30 C. ina 5-liter jar fermentor according to the same method described inexample 1. Recovery and purification of the product are conducted by thesame procedure as in example 2. As a result, 6.3 g. of a sugar-estermixture with the ester bond of trehalose as the constituent sugar and 2moles of arthronic acid is obtained.

EXAMPLE 4 Mycobacterium sp. ATCC 12297 is used as the seed microorganismand it is cultured in a seed medium prepared by adding 5 percent ofn-paraffins to the composition of the seed medium described in example2. The seed culture is conducted with aerobic shaking for 24 hours. Theresultant seed culture is inoculated into the same fermentation mediumand is cultured under the same conditions as described in example 1.After the completion of fermentation after 4 days, 2.3 g. of the esterof trehalose with a monocarboxylic acid, having the experimental formulaC H O is recovered from 1.6 liters of the fermentation liquor.

EXAMPLE 5 Arthrobacter mseoparajfinus ATCC 15584 is cultured for 72hours in the same manner as described in example 3. Separated from theresultant fermentation liquor are 1.2 g./1. of stearic acid diester oftrehalose, 0.3 g./1. of the arthronic acid monoester of glucose and 1.3g. of a mixture of stearic acid monoand tri-esters of sucrose.

EXAMPLE 6 Example 4 is repeated except that Micrococcus paraffinolylicusATCC 15582 is used as a seed strain in place of Mycobacterium sp. ATCC12297, whereby 3.5 g. of the ester of trehalose with a monocarboxylicacid, having the experimental formula C H O is recovered from 1.6 litersof the resultant fermentation liquor.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications as would be obvious to one skilled in the art are intendedto be included herein.

We claim:

1. A process for producing fatty acid esters of glucose, trehalose orsucrose which comprises culturing a hydrocarbon-assimilatingmicro-organism capable of producing said esters and belonging to a genusselected from the group consisting of Arthrobacter, Brevibacterium,Micrococcus, Corynebacterium, Mycobacterium, Candida and Aspergillusunder aerobic conditions in an aqueous nutrient medium containing ahydrocarbon or a mixture of hydrocarbons as the main carbon source,accumulating the fatty acid esters in the resultant culture liquor andrecovering said fatty acid esters therefrom.

2. The process of claim 1 wherein said hydrocarbon is an nparaffinhaving from six to 25 carbon atoms.

3. The process of claim 1 wherein said hydrocarbon is kerosene.

4. The process of claim 1, wherein culturing is carried out at atemperature of about 25 to 40 C. and at a pH of about 4 to 9.

5. A process for producing fatty acid esters of glucose, trehalose orsucrose which comprises culturing a hydrocarbon-assimilatingmicro-organism capable of producing said esters and belonging to a genusselected from the group eonsisting of Arthrobacter, Brevibacterium,Micrococcus, Corynebacterium, Mycobacterium, Candida and Aspergillusunder aerobic conditions at a temperature of about 25 to 40 C. and at apH of about 4 to 9 in an aqueous nutrient medium containing ahydrocarbon or a mixture of hydrocarbons as the main carbon source,accumulating the fatty acid esters in the resultant culture liquor, andrecovering said fatty acid esters therefrom.

6. The process of claim 5, wherein said hydrocarbon is an nparaffinhaving from six to 25 carbon atoms.

7. The process of claim 5 wherein said hydrocarbon is an nparafiinhaving from 1 1 to 18 carbon atoms.

8. The process of claim 5, wherein said hydrocarbon is kerosene.

9. The process of claim 5, wherein said fatty acid esters are recoveredfrom the oil layer part of the resultant fermentation liquor.

10. The process of claim 5, wherein said micro-organism isArthrobacterparaffineus ATCC 15591.

11. The process of claim 5, wherein said micro-organism isCorynebacterium hydrocarboclastus ATCC 15592.

12. The process of claim 5, wherein said micro-organism is Micrococcusepidermidis ATCC 155..

13. The process of claim 5, wherein said micro-organism is Mycobacteriumsp. ATCC 12297.

14. The process of claim 5, wherein said micro-organism is Arthrobacterroseoparaflinus ATCC 15584.

15. The process of claim 5, wherein said micro-organism is Micrococcusparaffinolyticus ATCC 15582.

2. The process of claim 1, wherein said hydrocarbon is an n-paraffinhaving from six to 25 carbon atoms.
 3. The process of claim 1, whereinsaid hydrocarbon is kerosene.
 4. The process of claim 1, whereinculturing is carried out at a temperature of about 25* to 40* C. and ata pH of about 4 to
 9. 5. A process for producing fatty acid esters ofglucose, trehalose or sucrose which comprises culturing ahydrocarbon-assimilating micro-organism capable of producing said estersand belonging to a genus selected from the group consisting ofArthrobacter, Brevibacterium, Micrococcus, Corynebacterium,Mycobacterium, Candida and Aspergillus under aerobic conditions at atemperature of about 25* to 40* C. and at a pH of about 4 to 9 in anaqueous nutrient medium containing a hydrocarbon or a mixture ofhydrocarbons as the main carbon source, accumulating the fatty acidesters in the resultant culture liquor, and recovering said fatty acidesters therefrom.
 6. The process of claim 5, wherein said hydrocarbon isan n-paraffin having from six to 25 carbon atoms.
 7. The process ofclaim 5, wherein said hydrocarbon is an n-paraffin having from 11 to 18carbon atoms.
 8. The process of claim 5, wherein said hydrocarbon iskerosene.
 9. The process of claim 5, wherein said fatty acid esters arerecovered from the oil layer part of the resultant fermentation liquor.10. The process of claim 5, wherein said micro-organism is Arthrobacterparaffineus ATCC
 15591. 11. The process of claim 5, wherein saidmicro-organism is Corynebacterium hydrocarboclastus ATCC
 15592. 12. Theprocess of claim 5, wherein said micro-organism is Micrococcusepidermidis ATCC
 155. 13. The process of claim 5, wherein saidmicro-organism is Mycobacterium sp. ATCC
 12297. 14. The process of claim5, wherein said micro-organism is Arthrobacter roseoparaffinus ATCC15584.
 15. The process of claim 5, wherein said micro-organism isMicrococcus paraffinolyticus ATCC 15582.